This invention relates to viral vascular endothelial growth factor-like proteins which binds and activates the mammalian VEGF receptor-2 to induce vascular permeability of endothelial cells, and in particular to pharmaceutical and diagnostic compositions and methods utilizing or derived from the factor.
In the developing embryo, the primary vascular network is established by in situ differentiation of mesodermal cells in a process called vasculogenesis. It is believed that all subsequent processes involving the generation of new vessels in the embryo and neovascularization in adults, are governed by the sprouting or splitting of new capillaries from the pre-existing vasculature in a process called angiogenesis (Pepper et al., Enzyme and Protein, 1996 49 138-162; Breier et al., Dev. Dyn. 1995 204 228-239; Risau, Nature, 1997 386 671-674). Angiogenesis is not only involved in embryonic development and normal tissue growth, repair, and regeneration, but is also involved in the female reproductive cycle, establishment and maintenance of pregnancy, and in repair of wounds and fractures. In addition to angiogenesis which takes place in the normal individual, angiogenic events are involved in a number of pathological processes, notably tumor growth and metastasis, and other conditions in which blood vessel proliferation, especially of the microvascular system, is increased, such as diabetic retinopathy, psoriasis and arthropathies. Inhibition of angiogenesis is useful in preventing or alleviating these pathological processes.
On the other hand, promotion of angiogenesis is desirable in situations where vascularization is to be established or extended, for example after tissue or organ transplantation, or to stimulate establishment of collateral circulation in tissue infarction or arterial stenosis, such as in coronary heart disease and thromboangitis obliterans.
The angiogenic process is highly complex and involves the maintenance of the endothelial cells in the cell cycle, degradation of the extracellular matrix, migration and invasion of the surrounding tissue and finally, tube formation. The molecular mechanisms underlying the complex angiogenic processes are far from being understood.
Because of the crucial role of angiogenesis in so many physiological and pathological processes, factors involved in the control of angiogenesis have been intensively investigated. A number of growth factors have been shown to be involved in the regulation of angiogenesis; these include fibroblast growth factors (FGFs), platelet-derived growth factor (PDGF), transforming growth factor alpha (TGFxcex1), and hepatocyte growth factor (HGF). See for example Folkman et al., J. Biol. Chem., 1992 267 10931-10934 for a review.
It has been suggested that a particular family of endothelial cell-specific growth factors, the vascular endothelial growth factors (VEGFs), and their corresponding receptors is primarily responsible for stimulation of endothelial cell growth and differentiation, and for certain functions of the differentiated cells. These factors are members of the PDGF family, and appear to act primarily via endothelial receptor tyrosine kinases (RTKs).
Nine different proteins have been identified in the PDGF family, namely two PDGFs (A and B), VEGF and six members that are closely related to VEGF. The six members closely related to VEGF are: VEGF-B, described in International Patent Application PCT/US96/02957 (WO 96/26736) and in U.S. Pat. Nos. 5,840,693 and 5,607,918 by Ludwig Institute for Cancer Research and The University of Helsinki; VEGF-C, described in Joukov et al., EMBO J., 1996 15 290-298 and Lee et al., Proc. Natl. Acad. Sci. USA, 1996 93 1988-1992; VEGF-D, described in International Patent Application No. PCT/US97/14696 (WO 98/07832), and Achen et al., Proc. Natl. Acad. Sci. USA, 1998 95 548-553; the placenta growth factor (PlGF), described in Maglione et al., Proc. Natl. Acad. Sci. USA, 1991 88 9267-9271; VEGF2, described in International Patent Application No. PCT/US94/05291 (WO 95/24473) by Human Genome Sciences, Inc; and VEGF3, described in International Patent Application No. PCT/US95/07283 (WO 96/39421) by Human Genome Sciences, Inc. Each VEGF family member has between 30% and 45% amino acid sequence identity with VEGF. The VEGF family members share a VEGF homology domain which contains the six cysteine residues which form the cysteine knot motif. Functional characteristics of the VEGF family include varying degrees of mitogenicity for endothelial cells, induction of vascular permeability and angiogenic and lymphangiogenic properties.
Vascular endothelial growth factor (VEGF) is a homodimeric glycoprotein that has been isolated from several sources. VEGF shows highly specific mitogenic activity for endothelial cells. VEGF has important regulatory functions in the formation of new blood vessels during embryonic vasculogenesis and in angiogenesis during adult life (Carmeliet et al., Nature, 1996 380 435-439; Ferrara et al., Nature, 1996 380 439-442; reviewed in Ferrara and Davis-Smyth, Endocrine Rev., 1997 18 4-25). The significance of the role played by VEGF has been demonstrated in studies showing that inactivation of a single VEGF allele results in embryonic lethality due to failed development of the vasculature (Carmeliet et al., Nature, 1996 380 435-439; Ferrara et al., Nature, 1996 380 439-442). In addition VEGF has strong chemoattractant activity towards monocytes, can induce the plasminogen activator and the plasminogen activator inhibitor in endothelial cells, and can also induce microvascular permeability. Because of the latter activity, it is sometimes referred to as vascular permeability factor (VPF). The isolation and properties of VEGF have been reviewed; see Ferrara et al., J. Cellular Biochem., 1991 47 211-218 and Connolly, J. Cellular Biochem., 1991 47 219-223. Alterative mRNA splicing of a single VEGF gene gives rise to five isoforms of VEGF.
VEGF-B has similar angiogenic and other properties to those of VEGF, but is distributed and expressed in tissues differently from VEGF. In particular, VEGF-B is very strongly expressed in heart, and only weakly in lung, whereas the reverse is the case for VEGF. This suggests that VEGF and VEGF-B, despite the fact that they are co-expressed in many tissues, may have functional differences.
VEGF-B was isolated using a yeast co-hybrid interaction trap screening technique by screening for cellular proteins which might interact with cellular resinoid acid-binding protein type I (CRABP-I). Its isolation and characteristics are described in detail in PCT/US96/02957 and in olofsson et al., Proc. Natl. Acad. Sci. USA, 1996 93 2576-2581.
VEGF-C was isolated from conditioned media of the PC-3 prostate adenocarcinoma cell line (CRL1435) by screening for ability of the medium to produce tyrosine phosphorylation of the endothelial cell-specific receptor tyrosine kinase VEGFR-3 (Flt4), using cells transfected to express VEGFR-3. VEGF-C was purified using affinity chromatography with recombinant VEGFR-3, and was cloned from a PC-3 cDNA library. Its isolation and characteristics are described in detail in Joukov et al., EMBO J., 1996 15 290-298.
VEGF-D was isolated from a human breast cDNA library, commercially available from Clontech, by screening with an expressed sequence tag obtained from a human cDNA library designated xe2x80x9cSoares Breast 3NbHBstxe2x80x9d as a hybridization probe (Achen et al., Proc. Natl. Acad. Sci. USA, 1998 95 548-553). Its isolation and characteristics are described in detail in International Patent Application No. PCT/US97/14696 (WO98/07832)
The VEGF-D gene is broadly expressed in the adult human, but is certainly not ubiquitously expressed. VEGF-D is strongly expressed in heart, lung and skeletal muscle. Intermediate levels of VEGF-D are expressed in spleen, ovary, small intestine and colon, and a lower expression occurs in kidney, pancreas, thymus, prostate and testis. No VEGF-D mRNA was detected in RNA from brain, placenta, liver or peripheral blood leukocytes.
PlGF was isolated from a term placenta cDNA library. Its isolation and characteristics are described in detail in Maglione et al., Proc. Natl. Acad. Sci. USA, 1991 88 9267-9271. Presently its biological function is not well understood.
VEGF2 was isolated from a highly tumorgenic, oestrogen-independent human breast cancer cell line. While this molecule is stated to have about 22% homology to PDGF and 30% homology to VEGF, the method of isolation of the gene encoding VEGF2 is unclear, and no characterization of the biological activity is disclosed.
VEGF3 was isolated from a cDNA library derived from colon tissue. VEGF3 is stated to have about 36% identity and 66% similarity to VEGF. The method of isolation of the gene encoding VEGF3 is unclear and no characterization of the biological activity is disclosed.
Similarity between two proteins is determined by comparing the amino acid sequence and conserved amino acid substitutions of one of the proteins to the sequence of the second protein, whereas identity is determined without including the conserved amino acid substitutions.
PDGF/VEGF family members act primarily by binding to receptor tyrosine kinases. Five endothelial cell-specific receptor tyrosine kinases have been identified, namely VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1), VEGFR-3 (Flt4), Tie and Tek/Tie-2. All of these have the intrinsic tyrosine kinase activity which is necessary for signal transduction. The essential, specific role in vasculogenesis and angiogenesis of VEGFR-1, VEGFR-2, VEGFR-3, Tie and Tek/Tie-2 has been demonstrated by targeted mutations inactivating these receptors in mouse embryos.
The only receptor tyrosine kinases known to bind VEGFs are VEGFR-1, VEGFR-2 and VEGFR-3. VEGFR-1 and VEGFR-2 bind VEGF with high affinity, and VEGFR-1 also binds VEGF-B and PlGF. VEGF-C has been shown to be the ligand for VEGFR-3, and it also activates VEGFR-2 (Joukov et al., The EMBO Journal, 1996 15 290-298). VEGF-D binds to both VEGFR-2 and VEGFR-3. A ligand for Tek/Tie-2 has been described in International Patent Application No. PCT/US95/12935 (WO 96/11269) by Regeneron Pharmaceuticals, Inc. The ligand for Tie has not yet been identified.
Recently, a novel 130-135 kDa VEGF isoform specific receptor has been purified and cloned (Soker et al., Cell, 1998 92 735-745). The VEGF receptor was found to specifically bind the VEGF165 isoform via the exon 7 encoded sequence, which shows weak affinity for heparin (Soker et al., Cell, 1998 92 735-745). Surprisingly, the receptor was shown to be identical to human neuropilin-1 (NP-1), a receptor involved in early stage neuromorphogenesis. PlGF-2 also appears to interact with NP-1 (Migdal et al., J. Biol. Chem., 1998 273 22272-22278).
VEGFR-1, VEGFR-2 and VEGFR-3 are expressed differently by endothelial cells. Both VEGFR-1 and VEGFR-2 are expressed in blood vessel endothelia (Oelrichs et al., Oncogene, 1992 8 11-18; Kaipainen et al., J. Exp. Med., 1993 178 2077-2088; Dumont et al., Dev. Dyn., 1995 203 80-92; Fong et al., Dev. Dyn., 1996 207 1-10) and VEGFR-3 is mostly expressed in the lymphatic endothelium of adult tissues (Kaipainen et al., Proc. Natl. Acad. Sci. USA, 1995 9 3566-3570). VEGFR-3 is also expressed in the blood vasculature surrounding tumors.
Disruption of the VEGFR genes results in aberrant development of the vasculature leading to embryonic lethality around midgestation. Analysis of embryos carrying a completely inactivated VEGFR-1 gene suggests that this receptor is required for functional organization of the endothelium (Fong et al., Nature, 1995 376 66-70). However, deletion of the intracellular tyrosine kinase domain of VEGFR-1 generates viable mice with a normal vasculature (Hiratsuka et al., Proc. Natl. Acad. Sci. USA 1998 95 9349-9354). The reasons underlying these differences remain to be explained but suggest that receptor signalling via the tyrosine kinase is not required for the proper function of VEGFR-1. Analysis of homozygous mice with inactivated alleles of VEGFR-2 suggests that this receptor is required for endothelial cell proliferation, hematopoesis and vasculogenesis (Shalaby et al., Nature, 1995 376 62-66; Shalaby et al., Cell, 1997 89 981-990). Inactivation of VEGFR-3 results in cardiovascular failure due to abnormal organization of the large vessels (Dumont et al. Science, 1998 282 946-949).
Although VEGFR-1 is mainly expressed in endothelial cells during development, it can also be found in hematopoetic precursor cells during early stages of embryogenesis (Fong et al., Nature, 1995 376 66-70). In adults, monocytes and macrophages also express this receptor (Barleon et al., Blood, 1996 87 3336-3343). In embryos, VEGFR-1 is expressed by most, if not all, vessels (Breier et al., Dev. Dyn., 1995 204 228-239; Fong et al., Dev. Dyn., 1996 207 1-10).
The receptor VEGFR-3 is widely expressed on endothelial cells during early embryonic development but as embryogenesis proceeds becomes restricted to venous endothelium and then to the lymphatic endothelium (Kaipainen et al., Cancer Res., 1994 54 6571-6577; Kaipainen et al., Proc. Natl. Acad. Sci. USA, 1995 92 3566-3570). VEGFR-3 is expressed on lymphatic endothelial cells in adult tissues. This receptor is essential for vascular development during embryogenesis. Targeted inactivation of both copies of the VEGFR-3 gene in mice resulted in defective blood vessel formation characterized by abnormally organized large vessels with defective lumens, leading to fluid accumulation in the pericardial cavity and cardiovascular failure at post-coital day 9.5. On the basis of these findings it has been proposed that VEGFR-3 is required for the maturation of primary vascular networks into larger blood vessels. However, the role of VEGFR-3 in the development of the lymphatic vasculature could not be studied in these mice because the embryos died before the lymphatic system emerged. Nevertheless it is assumed that VEGFR-3 plays a role in development of the lymphatic vasculature and lymphangiogenesis given its specific expression in lymphatic endothelial cells during embryogenesis and adult life. This is supported by the finding that ectopic expression of VEGF-C, a ligand for VEGFR-3, in the skin of transgenic mice, resulted in lymphatic endothelial cell proliferation and vessel enlargement in the dermis. Furthermore this suggests that VEGF-C may have a primary function in lymphatic endothelium, and a secondary function in angiogenesis and permeability regulation which is shared with VEGF (Joukov et al., EMBO J., 1996 15 290-298).
Some inhibitors of the VEGF/VEGF-receptor system have been shown to prevent tumor growth via an anti-angiogenic mechanism; see Kim et al., Nature, 1993 362 841-844 and Saleh et al., Cancer Res., 1996 56 393-401.
In addition, VEGF-like proteins have been identified which are encoded by four different strains of the orf virus. This is the first virus reported to encode a VEGF-like protein. The first two strains are NZ2 and NZ7, and are described in Lyttle et al., J. Virol., 1994 68 84-92. A third is D1701 and is described in Meyer et al., The EMBO Journal, 1999 18 363-374. The fourth strain is NZ10 and is described herein. These proteins show amino acid sequence similarity to VEGF and to each other.
The orf virus is a type of species of the parapoxvirus genus which causes a highly contagious pustular dermatitis in sheep and goats and is readily transmittable to humans. The pustular dermatitis induced by orf virus infection is characterized by dilation of blood vessels, swelling of the local area and marked proliferation of endothelial cells lining the blood vessels. These features are seen in all species infected by orf and can result in the formation of a tumor-like growth or nodule due to viral replication in epidermal cells. Generally orf virus infections resolve in a few weeks but severe infections that fail to resolve without surgical intervention are seen in immune impaired individuals. The finding that the orf virus strains NZ2 and NZ7 encode molecules with VEGF-like sequences raises the important question of whether these proteins are capable of binding to mammalian VEGF receptors and inducing characteristic VEGF-like effects such as mitogenesis of endothelial cells and vascular permeability.
The invention is based on the discovery that a viral VEGF-like protein from the orf virus strains NZ2 (herein after referred to as ORFV2-VEGF or NZ2)and NZ10 are capable of binding to the extracellular domain of the VEGF receptor-2 to form bioactive complexes which mediate useful cellular responses and/or antagonize undesired biological activities. In addition, ORFV2-VEGF is capable of binding NP-1. The invention generally provides for methods which stimulate or inhibit these biological activities, methods for therapeutic applications and finding antagonists of ORFV2-VEGF or NZ10.
According to a first aspect, the invention provides an isolated and purified nucleic acid molecule which comprises a polynucleotide sequence having at least 85% identity, more preferably at least 90%, and most preferably at least 95% identity to at least the sequence set out in FIG. 10 (SEQ ID NO:10) and that encodes a novel polypeptide, designated ORFV10-VEGF (hereinafter NZ10), which is structurally homologous to VEGF and NZ2. This aspect of the invention also encompasses DNA molecules having a sequence such that they hybridize under stringent conditions with at least nucleotides of the sequence set out in FIG. 10 (SEQ ID NO:10) or fragments thereof.
According to a second aspect, the polypeptide of the invention has the ability to stimulate proliferation of endothelial cells and comprises a sequence of amino acids substantially corresponding to the amino acid sequence set out in FIG. 11 (SEQ ID NO:11), or a fragment or analog thereof which has the ability to stimulate one or more of endothelial cell proliferation, differentiation, migration or survival. Preferably the polypeptides have at least 85% identity, more preferably at least 90%, and most preferably at least 95% identity to the amino acid sequence of FIG. 11 (SEQ ID NO:11), or a fragment or analog thereof having the biological activity of NZ10.
According to another aspect, the invention provides for a method for stimulating one or more of endothelial cell proliferation, differentiation, migration or survival by exposing them to ORFV2-VEGF or NZ10 or a fragment or analog thereof which has the ability.
According to a further aspect, the invention provides a method for activation of VEGF receptor-2 which comprises the step of exposing cells bearing said receptor to an effective receptor activating dose of ORFV2-VEGF or NZ10 or a fragment or analog thereof which has the ability.
Since both ORFV2-VEGF and NZ10 specifically activate the VEGF receptor-2, ORFV2-VEGF can be used to stimulate endothelial cell proliferation in a situation where VEGF receptor 1 is not activated. Accordingly, the invention provides for a method for specific activation of VEGF receptor 2 and VEGF receptor 1 is not activated.
These abilities are referred to herein as xe2x80x9cbiological activities of ORFV2-VEGF or NZ10xe2x80x9d and can readily be tested by methods known in the art, such as the mitogenic assay described in Example 5. In particular, ORFV2-VEGF and NZ10 have the ability to stimulate endothelial cell proliferation or differentiation, including, but not limited to, proliferation or differentiation of vascular endothelial cells and/or lymphatic endothelial cells.
More preferably ORFV2-VEGF has the amino acid sequence set out in FIG. 9 (SEQ ID NO:2), while NZ10 has the amino acid sequence set out in FIG. 10 (SEQ ID NO:11).
In another preferred aspect, the invention provides a polypeptides possessing the characteristic amino acid sequence:
Pro-Xaa-Cys-Xaa-Xaa-Xaa-Xaa-Arg-Cys-Xaa-Gly-Cys-Cys
(SEQ ID NOs:9 and 11), which is characteristic of members of the PDGF/VEGF family of growth factors.
Polypeptides comprising conservative substitutions, insertions, or deletions, but which still retain the biological activity of ORFV2-VEGF or NZ10, are clearly to be understood to be within the scope of the invention. Persons skilled in the art will be well aware of methods which can readily be used to generate such polypeptides, for example the use of site-directed mutagenesis, or specific enzymatic cleavage and ligation. The skilled person will also be aware that peptidomimetic compounds or compounds in which one or more amino acid residues are replaced by a non-naturally occurring amino acid or an amino acid analogue may retain the required aspects of the biological activity of ORFV2-VEGF. Such compounds can readily be made and tested by methods known in the art, and are also within the scope of the invention.
In addition, variant forms of the ORFV2-VEGF or NZ10 polypeptide which result naturally-occurring allelic variants of the nucleic acid sequence encoding ORFV2-VEGF or ORFV10-VEGF are encompassed within the scope of the invention. Allelic variants are well known in the art, and represent alternative forms or a nucleic acid sequence which comprise substitution, deletion or addition of one or more nucleotides, but which do not result in any substantial functional alteration of the encoded polypeptide.
As used herein, the term xe2x80x9cORFV2-VEGFxe2x80x9d collectively refers to the polypeptide having the amino acid sequence set forth in FIG. 9 (SEQ ID NO:2) and fragments or analogues thereof and other variants, for example, from natural isolates of the orf virus which have the biological activity of ORFV2-VEGF as herein defined. Those skilled in the art will recognize that there is considerable latitude in amino acid sequence charges which can occur naturally or be engineered without affecting biological activity of the polypeptide. It is preferred that the variant polypeptides be at least 80%, more preferably be at least 90%, and most preferably at least 95% identical to the amino acid sequence of FIG. 9 (SEQ ID NO:2). Percent sequence identity is determined by conventional methods. See, for example, Altschul et al, Bull. Math. Bio., 1986 48 603-616 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 1992 89 10915-10919.
As used herein, the term xe2x80x9cNZ10xe2x80x9d collectively refers to the polypeptide having the amino acid sequence set forth in FIG. 11 (SEQ ID NO:11) and fragments or analogs thereof and other variants, for example, from natural isolates of the orf virus which have the biological activity of NZ10 as herein defined. Those skilled in the art will recognize that there is considerable latitude in amino acid sequence charges which can occur naturally or be engineered without affecting biological activity of the polypeptide. It is preferred that the variant polypeptides be at least 80%, more preferably be at least 90%, and most preferably at least 95% identical to the amino acid sequence of FIG. 11 (SEQ ID NO:11). Percent sequence identity is determined by conventional methods. See, for example, Altschul et al, Bull. Math. Bio., 1986 48 603-616 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 1992 89 10915-10919.
Such variant forms of ORFV2-VEGF or NZ10 can be prepared by targeting non-essential regions of the ORFV2-VEGF or NZ10 polypeptide for modification. Other variant forms may be naturally made from related orf virus strains. These non-essential regions are expected to fall outside the strongly-conserved regions indicated in FIGS. 1A and 1B. In particular, the growth factors of the PDGF family, including VEGF, are dimeric, and VEGF, VEGF-B, VEGF-C, VEGF-D, ORFV2-VEGF, PlGF, PDGF-A and PDGF-B show complete conservation of eight cysteine residues in the N-terminal domains, ie. the PDGF-like domains (Olofsson et al, 1996; Joukov et al, 1996). These cysteines are thought to be involved in intra- and inter-molecular disulfide bonding. In addition there are further strongly, but not completely, conserved cysteine residues in the C-terminal domains. Loops 1, 2 and 3 of each subunit, which are formed by intra-molecular disulfide bonding, are involved in binding to the receptors for the PDGF/VEGF family of growth factors (Andersson et al: Growth Factors, 1995 12 159-164). As noted above, the cysteines conserved in previously known members of the VEGF family are also conserved in ORFV2-VEGF.
Persons skilled in the art thus are well aware that these cysteine residues should be preserved in any proposed variant form, and that the active sites present in loops 1, 2 and 3 also should be preserved. However, other regions of the molecule can be expected to be of lesser importance for biological function, and therefore offer suitable targets for modification. Modified polypeptides can readily be tested for their ability to show the biological activity of ORFV2-VEGF by routine activity assay procedures such as cell proliferation tests.
It is contemplated that some modified ORFV2-VEGF or NZ10 polypeptides will have the ability to bind to endothelial cells, e.g. to VEGF receptor-2, but will be unable to stimulate endothelial cell proliferation, differentiation, migration or survival. These modified polypeptides are expected to be able to act as competitive or non-competitive inhibitors of the ORFV2-VEGF or NZ10 polypeptides and growth factors of the PDGF/VEGF family, and to be useful in situations where prevention or reduction of the ORFV2-VEGF or NZ10 polypeptide or PDGF/VEGF family growth factor action is desirable. Thus such receptor-binding but non-mitogenic, non-differentiation inducing, non-migration inducing, non-motility inducing, non-survival promoting, non-connective tissue development promoting, non-wound healing or non-vascular proliferation inducing variants of the ORFV2-VEGF or NZ10 polypeptide are also within the scope of the invention, and are referred to herein as xe2x80x9creceptor-binding but otherwise inactive variantxe2x80x9d. Because ORFV2-VEGF or NZ10 forms a dimer in order to activate its only known receptor, it is contemplated that one monomer comprises the receptor-binding but otherwise inactive variant modified ORFV2-VEGF or NZ10 polypeptide and a second monomer comprises a wild-type ORFV2-VEGF or NZ10 or a wild-type growth factor of the PDGF/VEGF family. These dimers can bind to its corresponding receptor but cannot induce downstream signaling.
It is also contemplated that there are other modified ORFV2-VEGF or NZ10 polypeptides that can prevent binding of a wild-type ORFV2-VEGF or NZ10 or a wild-type growth factor of the PDGF/VEGF family to its corresponding receptor on endothelial cells. Thus these dimers will be unable to stimulate endothelial cell proliferation, differentiation, migration or survival. These modified polypeptides are expected to be able to act as competitive or non-competitive inhibitors of the ORFV2-VEGF or NZ10 polypeptide or a growth factor of the PDGF/VEGF family, and to be useful in situations where prevention or reduction of the ORFV2-VEGF or NZ10 polypeptide or PDGF/VEGF family growth factor action is desirable. Such situations include the tissue remodeling that takes place during invasion of tumor cells into a normal cell population by primary or metastatic tumor formation. Thus such the ORFV2-VEGF or NZ10 or PDGF/VEGF family growth factor-binding but non-mitogenic, non-differentiation inducing, non-migration inducing, non-motility inducing, non-survival promoting, non-connective tissue promoting, non-wound healing or non-vascular proliferation inducing variants of the ORFV2-VEGF or NZ10 polypeptide are also within the scope of the invention, and are referred to herein as xe2x80x9cthe ORFV2-VEGF or NZ10 polypeptide-dimer forming but otherwise inactive or interfering variantsxe2x80x9d.
Thus, another aspect of the invention is a ORFV2-VEGF or NZ10 antagonist, wherein the antagonist is an isolated polypeptide which comprises a sequence of amino acids substantially corresponding to the amino acid sequence of FIG. 9 (SEQ ID NO:2) or FIG. 11 (SEQ ID NO:11), repsectively and has the ability to bind to ORFV2-VEGF or NZ10 and to prevent biological activity of ORFV2-VEGF or NZ10.
As noted above, the orf virus is known to cause a pustular dermatitis in sheep, goats and humans. The lesions induced after infection with orf virus show extensive proliferation of vascular endothelial cells, dilation of blood vessels and dermal swelling. Expression of an orf virus gene able to stimulate angiogenesis may provide an explanation for these histological observations.
Accordingly, a further aspect of the invention provides a method for treatment of pustular dermatitis and of fluid accumulation caused by viral infection which comprises the step of administering a therapeutically effective amount of an antagonist to ORFV2-VEGF or NZ10 or to the VEGF receptor 2.
Where ORFV2-VEGF, NZ10 or a ORFV2-VEGF antagonist or NZ10 antagonist is to be used for therapeutic purposes, the dose and route of application will depend upon the condition to be treated, and will be at the discretion of the attending physician or veterinarian. Suitable routes include subcutaneous, intramuscular, intraperitoneal or intravenous injection, topical application, implants etc. Topical application of ORFV2-VEGF or NZ10 may be used in a manner analogous to VEGF.
Another aspect of the invention provides expression vectors comprising the DNA of the invention or a nucleic acid molecule of the invention, and host cells transformed or transfected with nucleic acids molecules or vectors of the invention. Vectors also comprises a nucleic acid sequence which hybridize under stringent conditions with the sequence of FIG. 8 or FIG. 10. These cells are particularly suitable for expression of the polypeptide of the invention, and include insect cells such as Sf9 cells, obtainable from the American Type Culture Collection (ATCC SRL-171), transformed with a baculovirus vector, and the human embryo kidney cell line 293 EBNA transfected by a suitable expression plasmid. Preferred vectors of the invention are expression vectors in which a nucleic acid according to the invention is operatively connected to one or more appropriate promoters and/or other control sequences, such that appropriate host cells transformed or transfected with the vectors are capable of expressing the polypeptide of the invention. Other preferred vectors are those suitable for transfection of mammalian cells, or for gene therapy, such as adenoviral-, vaccinia- or retroviral-based vectors or liposomes. A variety of such vectors is known in the art.
The invention also provides a method of making a vector capable of expressing a polypeptide encoded by a nucleic acid according to the invention, comprising the steps of operatively connecting the nucleic acid to one or more appropriate promoters and/or other control sequences, as described above.
The invention further provides a method of making a polypeptide according to the invention, comprising the steps of expressing a nucleic acid or vector according to the invention in a host cell, and isolating the polypeptide from the host cell or from the host cell""s growth medium.
In yet a further aspect, the invention provides an antibody specifically reactive with ORFV2-VEGF or NZ10. This aspect of the invention includes antibodies specific for the variant forms, fragments and analogues of ORFV2-VEGF or NZ10 referred to above. The term xe2x80x9canalogxe2x80x9d or xe2x80x9cfunctional analogxe2x80x9d refers to a modified form of ORFV2-VEGF or NZ10 in which at least one amino acid substitution has been made such that said analog retains substantially the same biological activity as the unmodified ORFV2-VEGF and/or NZ10 in vivo and or in vitro. Such antibodies are useful as inhibitors or agonists of ORFV2-VEGF or NZ10 and as diagnostic agents for detecting and quantifying ORFV2-VEGF and/or NZ10. Polyclonal or monoclonal antibodies may be used. Monoclonal and polyclonal antibodies can be raised against polypeptides of the invention using standard methods in the art. For some purposes, for example where a monoclonal antibody is to be used to inhibit effects of ORFV2-VEGF and/or NZ10 in a clinical situation, it may be desirable to use humanized or chimeric monoclonal antibodies. In addition the polypeptide can be linked to an epitope tag, such as the FLAG(copyright) octapeptide (Sigma, St. Louis, Mo.), to assist in affinity purification. For some purposes, for example where a monoclonal antibody is to be used to inhibit effects of PDGF-C in a clinical situation, it may be desirable to use humanized or chimeric monoclonal antibodies. Such antibodies may be further modified by addition of cytotoxic or cytostatic drugs. Methods for producing these, including recombinant DNA methods, are also well known in the art.
This aspect of the invention also includes an antibody which recognizes ORFV2-VEGF and which is suitably labeled.
Polypeptides or antibodies according to the invention may be labeled with a detectable label, and utilized for diagnostic purposes. Similarly, the thus-labeled polypeptide of the invention may be used to identify its corresponding receptor in situ. The polypeptide or antibody may be covalently or non-covalently coupled to a suitable supermagnetic, paramagnetic, electron dense, ecogenic or radioactive agent for imaging. For use in diagnostic assays, radioactive or non-radioactive labels may be used. Examples of radioactive labels include a radioactive atom or group, such as 125 I or 32p. Examples of non-radioactive labels include enzymatic labels, such as horseradish peroxidase or fluorimetric labels, such as fluorescein-5-isothiocyanate (FITC). Labeling may be direct or indirect, covalent or non-covalent.
Clinical applications of the invention include diagnostic applications, acceleration of angiogenesis in wound healing, tissue or organ transplantation, or to establish collateral circulation in tissue infarction or arterial stenosis, such as coronary artery disease, and inhibition of angiogenesis in the treatment of cancer or of diabetic retinopathy.
Conversely, ORFV2-VEGF and/or NZ10 antagonists (e.g. antibodies and/or inhibitors) could be used to treat conditions, such as congestive heart failure, involving accumulations of fluid in, for example, the lung resulting from increases in vascular permeability, by exerting an offsetting effect on vascular permeability in order to counteract the fluid accumulation. Administrations of ORFV2-VEGF could be used to treat malabsorptive syndromes in the intestinal tract as a result of its blood circulation increasing and vascular permeability increasing activities.
Thus the invention provides a method of stimulation of angiogenesis and/or neovascularization in a mammal in need of such treatment, comprising the step of administering an effective dose of ORFV2-VEGF or NZ10, or a fragment or analog thereof which has the ability to stimulate endothelial cell proliferation, to the mammal.
Optionally ORFV2-VEGF may be administered together with, or in conjunction with, one or more of VEGF, VEGF-B, VEGF-C, VEGF-D, PlGF, PDGF, FGF and/or heparin.
Conversely the invention provides a method of inhibiting angiogenesis and/or neovascularization in a mammal in need of such treatment, comprising the step of administering an effective amount of an antagonist of ORFV2-VEGF to the mammal. The antagonist may be any agent that prevents the action of ORFV2-VEGF and/or NZ10, either by preventing the binding of ORFV2-VEGF and/or NZ10 to its corresponding receptor or the target cell, or by preventing activation of the transducer of the signal from the receptor to its cellular site of action. Suitable antagonists include, but are not limited to, antibodies directed against ORFV2-VEGF and/or NZ10; competitive or non-competitive inhibitors of binding of ORFV2-VEGF/NZ10 to the ORFV2-VEGF/NZ10 receptor, such as the receptor-binding but non-mitogenic ORFV2-VEGF or NZ10 variants referred to above; and anti-sense nucleotide sequences complementary to at least a part of the DNA sequence encoding ORFV2-VEGF and/or NZ10.
The invention also provides a method of detecting ORFV2-VEGF and/or NZ10 in a biological sample, comprising the step of contacting the sample with a reagent capable of binding ORFV2-VEGF, and detecting the binding. Preferably the reagent capable of binding ORFV2-VEGF and/or NZ10 is an antibody directed against ORFV2-VEGF and/or NZ10, particularly preferably a monoclonal antibody. In a preferred embodiment the binding and/or extent of binding is detected by means of a detectable label; suitable labels are discussed above.
According to yet a further aspect, the invention provides diagnostic means typically in the form of test kits. For example, in one embodiment of the invention there is provided a diagnostic test kit comprising an antibody to ORFV2-VEGF and/or NZ10 and means for detecting, and more preferably evaluating, binding between the antibody and ORFV2-VEGF or NZ10. In one preferred embodiment of the diagnostic means according to the invention, either the antibody or the ORFV2-VEGF or NZ10 is labelled with a detectable label, and either the antibody or the ORFV2-VEGF or NZ10 is substrate-bound, such that the ORFV2-VEGF/ or NZ10/antibody interaction can be established by determining the amount of label attached to the substrate following binding between the antibody and the ORFV2-VEGF and/or NZ10. In a particularly preferred embodiment of the invention, the diagnostic means may be provided as a conventional ELISA kit.
A method is provided for determining agents that bind to ORFV2-VEGF and/or NZ10. The method comprises contacting ORFV2-VEGF or NZ10 with a test agent and monitoring binding by any suitable means. Agents can include both compounds and other proteins.
The invention provides a screening system for discovering agents that bind ORFV2-VEGF and/or NZ10. The screening system comprises preparing ORFV2-VEGF or NZ10, exposing ORFV2-VEGF or NZ10 to a test agent, and quantifying the binding of said agent to ORFV2-VEGF or NZ10 by any suitable means.
Use of this screen system provides a means to determine compounds that may alter the biological function of ORFV2-VEGF or NZ10. This screening method may be adapted to large-scale, automated procedures such as a PANDEX(copyright) (Baxter-Dade Diagnostics) system, allowing for efficient high-volume screening of potential therapeutic agents.
For this screening system, ORFV2-VEGF or NZ10 is prepared as described herein, preferably using recombinant DNA technology. A test agent, e.g. a compound or protein, is introduced into a reaction vessel containing ORFV2-VEGF or NZ10. Binding of the test agent to ORFV2-VEGF or NZ10 is determined by any suitable means which include, but is not limited to, radioactively- or chemically-labeling the test agent. Binding of ORFV2-VEGF or NZ10 may also be carried out by a method disclosed in U.S. Pat. No. 5,585,277, which is incorporated by reference. In this method, binding of the test agent to ORFV2-VEGF or NZ10 is assessed by monitoring the ratio of folded protein to unfolded protein. Examples of this monitoring can include, but are not limited to, amenability to binding of the protein by a specific antibody against the folded state of the protein.
Those of skill in the art will recognize that IC50 values are dependent on the selectivity of the agent tested. For example, an agent with an IC50 which is less than 10 nM is generally considered an excellent candidate for drug therapy. However, an agent which has a lower affinity, but is selective for a particular target, may be an even better candidate. Those skilled in the art will recognize that any information regarding the binding potential, inhibitory activity or selectivity of a particular agent is useful toward the development of pharmaceutical products.
Where a ORFV2-VEGF or NZ10 or a ORFV2-VEGF antagonist or a NZ10 antagonist is to be used for therapeutic purposes, the dose(s) and route of administration will depend upon the nature of the patient and condition to be treated, and will be at the discretion of the attending physician or veterinarian. Suitable routes include oral, subcutaneous, intramuscular, intraperitoneal or intravenous injection, parenteral, topical application, implants etc. Topical application of ORFV2-VEGF or NZ10 may be used in a manner analogous to VEGF. For example, where used for wound healing or other use in which enhanced angiogenesis is advantageous, an effective amount of ORFV2-VEGF or NZ10 is administered to an organism in need thereof in a dose between about 0.1 and 1000 xcexcg/kg body weight.
The ORFV2-VEGF or NZ10 or a ORFV2-VEGF antagonist or a NZ10 antagonist may be employed in combination with a suitable pharmaceutical carrier. The resulting compositions comprise a therapeutically effective amount of ORFV2-VEGF or NZ10 or a ORFV2-VEGF antagonist or a NZ10 antagonist, and a pharmaceutically acceptable non-toxic salt thereof, and a pharmaceutically acceptable solid or liquid carrier or adjuvant. Examples of such a carrier or adjuvant include, but are not limited to, saline, buffered saline, Ringer""s solution, mineral oil, talc, corn starch, gelatin, lactose, sucrose, microcrystalline cellulose, kaolin, mannitol, dicalcium phosphate, sodium chloride, alginic acid, dextrose, water, glycerol, ethanol, thickeners, stabilizers, suspending agents and combinations thereof. Such compositions may be in the form of solutions, suspensions, tablets, capsules, creams, salves, elixirs, syrups, wafers, ointments or other conventional forms. The formulation to suit the mode of administration. Compositions which comprise ORFV2-VEGF or NZ10 may optionally further comprise one or more of PDGF-A, PDGF-B, VEGF, VEGF-B, VEGF-C, VEGF-D, PlGF and/or heparin. Compositions comprising ORFV2-VEGF or NZ10 will contain from about 0.1% to 90% by weight of the active compound(s), and most generally from about 10% to 30%.
For intramuscular preparations, a sterile formulation, preferably a suitable soluble salt form of ORFV2-VEGF or NZ10, such as hydrochloride salt, can be dissolved and administered in a pharmaceutical diluent such as pyrogen-free water (distilled), physiological saline or 5% glucose solution. A suitable insoluble form of the compound may be prepared and administered as a suspension in an aqueous base or a pharmaceutically acceptable oil base, e.g. an ester of a long chain fatty acid such as ethyl oleate.
Another aspect of the invention concerns the provision of a pharmaceutical composition comprising either ORFV2-VEGF or NZ10 or a fragment or analog thereof which promotes proliferation of endothelial cells, or an antagonist such as antibody thereto. Compositions which comprise ORFV2-VEGF or NZ10 may optionally further comprise one or more of VEGF, VEGF-B, VEGF-C, VEGF-D and/or heparin.
In another aspect, the invention relates to a protein dimer comprising ORFV2-VEGF or NZ10, particularly a disulfide-linked dimer. The protein dimers of the invention include both homodimers of ORFV2-VEGF or NZ10 and heterodimers of ORFV2-VEGF or NZ10 and VEGF, VEGF-B, VEGF-C, VEGF-D, PlGF or PDGF, or heterodimers of ORFV2-VEGF and NZ10.
According to a yet further aspect of the invention there is provided a ORFV2-VEGF and/or NZ10 antagonist which can be an anti-sense nucleotide sequence which is complementary to at least a part of a DNA sequence which encodes ORFV2-VEGF, NZ10 or a fragment or analog thereof which may be used to inhibit, or at least mitigate, ORFV2-VEGF and/or NZ10 expression. In addition an anti-sense nucleotide sequence can be to the promoter region of the ORVF2-VEGF or ORFV10-VEGF gene or other non-coding region of the gene which may be used to inhibit, or at least mitigate, ORFV2-VEGF and/or NZ10 expression. The use of an antagonist of this type to inhibit ORFV2-VEGF expression is favored in instances where ORFV2-VEGF expression is associated with a disease, for example pustular dermatitis. Transformation of such cells with a vector containing an anti-sense nucleotide sequence would suppress or retard angiogenesis, and so would inhibit or retard growth of lesions.
A still further aspect the invention relates to an isolated ORFV2-VEGF or NZ10 and VEGFR-2 complex. Isolation and purification of complexes could be effected by conventional procedures such as immunoaffinity purification using monoclonal antibodies according to techniques described in standard reference work such as Harlow et al, xe2x80x9cAntibodies, a Laboratory Manualxe2x80x9d, Cold Spring Harbor Laboratory Press, 1988 and/or Marshak et al, xe2x80x9cStrategies for Protein Purification and Characterizationxe2x80x9d, Cold Spring Harbor Laboratory Press, 1986.
Polynucleotides of the invention such as those described above, fragments of those polynucleotides, and variants of those polynucleotides with sufficient similarity to the non-coding strand of those polynucleotides to hybridize thereto under stringent conditions all are useful for identifying, purifying, and isolating polynucleotides encoding other, viral forms of VEGF-like polypeptides. Thus, such polynucleotide fragments and variants are intended as aspects of the invention. Exemplary stringent hybridization conditions are as follows: hybridization at 42xc2x0 C. in 5xc3x97SSC, 20 mM NaPO4, pH 6.8, 50% formamide; and washing at 42xc2x0 C. in 0.2xc3x97SSC. Those skilled in the art understand that it is desirable to vary these conditions empirically based on the length and the GC nucleotide base content of the sequences to be hybridized, and that formulas for determining such variation exist. See for example Sambrook et al, xe2x80x9cMolecular Cloning: A Laboratory Manualxe2x80x9d, Second Edition, pages 9.47-9.51, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory (1989).
It will be clearly understood that nucleic acids and polypeptides of the invention may be prepared by synthetic means or by recombinant means, or may be purified from natural sources.
It will be clearly understood that for the purposes of this specification the word xe2x80x9ccomprisingxe2x80x9d means xe2x80x9cincluded but not limited toxe2x80x9d. The corresponding meaning applies to the word xe2x80x9ccomprisesxe2x80x9d.